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It has been reported that, in the spontaneously hypertensive rat (SHR) model of hypertension, different components of the G-protein/adenylate cyclase (AC)/Calcium-activated potassium channel of high conductance (BK) channel signaling pathway are altered differently. In the upstream part of the pathway (G-protein/AC), a comparatively low efficacy has been established, whereas downstream BK currents seem to be increased. Thus, the overall performance of this signaling pathway in SHR is elusive. For a better understanding, we focused on one aspect, the direct targeting of the BK channel by the G-protein/AC pathway and tested the hypothesis that the comparatively low AC pathway efficacy in SHR results in a reduced agonist-induced stimulation of BK currents. This hypothesis was investigated using freshly isolated smooth muscle cells from WKY and SHR rat tail artery and the patch-clamp technique. It was observed that: (1) single BK channels have similar current-voltage relationships, voltage-dependence and calcium sensitivity; (2) BK currents in cells with a strong buffering of the BK channel activator calcium have similar current-voltage relationships; (3) the iloprost-induced concentration-dependent increase of the BK current is larger in WKY compared to SHR; (4) the effects of activators of the PKA pathway, the catalytic subunit of PKA and the potent and selective cAMP-analogue Sp-5,6-DCl-cBIMPS on BK currents are similar. Thus, our data suggest that the lower iloprost-induced stimulation of the BK current in freshly isolated rat tail artery smooth muscle cells from SHR compared with WKY is due to the lower efficacy of upstream elements of the G-Protein/AC/BK channel pathway.

Modafinil has been used as a psychostimulant for the treatment of narcolepsy. However, its primary mechanism of action remains elusive. Therefore, we examined the effects of modafinil on K(Ca)3.1 channels and vascular smooth muscle contraction. K(Ca)3.1 currents and channel activity were measured using a voltage-clamp technique and inside-out patches in mouse embryonic fibroblast cell line, NIH-3T3 fibroblasts. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration was measured, and the phosphorylation of K(Ca)3.1 channel protein was examined using western blotting in NIH-3T3 fibroblasts and/or primary cultured mouse aortic smooth muscle cells (SMCs). Muscle contractions were recorded from mouse aorta and rat pulmonary artery by using a myograph developed in-house. Modafinil was found to inhibit K(Ca)3.1 currents in a concentration-dependent manner, and the half-maximal inhibition (IC(50)) of modafinil for the current inhibition was 6.8 ± 0.7 nM. The protein kinase A (PKA) activator forskolin also inhibited K(Ca)3.1 currents. The inhibitory effects of modafinil and forskolin on K(Ca)3.1 currents were blocked by the PKA inhibitors PKI(14-22) or H-89. In addition, modafinil relaxed blood vessels (mouse aorta and rat pulmonary artery) in a concentration-dependent manner. Modafinil increased cAMP concentrations in NIH-3T3 fibroblasts or primary cultured mouse aortic SMCs and phosphorylated K(Ca)3.1 channel protein in NIH-3T3 fibroblasts. However, open probability and single-channel current amplitudes of K(Ca)3.1 channels were not changed by modafinil. From these results, we conclude that modafinil inhibits K(Ca)3.1 channels and vascular smooth muscle contraction by cAMP-dependent phosphorylation, suggesting that modafinil can be used as a cAMP-dependent K(Ca)3.1 channel blocker and vasodilator., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

As powerful vasodilators, prostacyclin analogues are presently the mainstay in the treatment of severe pulmonary arterial hypertension. Although the hemodynamic effects of prostacyclin analogues are well known, the molecular mechanism of their acute effects on pulmonary vascular tone and systemic vascular tone remains poorly understood. Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) was previously identified as a putative receptor responsible for the modulation of target gene expression in response to prostacyclin analogues. The present study investigated the signaling pathway of prostacyclin in human pulmonary arterial smooth muscle cells (PASMCs), and sought to define the role of PPARβ/δ in the acute vasodilating effect. In human PASMCs, prostacyclin rapidly activated TWIK-related acid-sensitive K channel 1 (TASK-1) and calcium-dependent potassium channels (K(Ca)). This pathway was mediated via the prostanoid I receptor-protein kinase A pathway. The silencing of PPARβ/δ demonstrated that the downstream K(Ca) activation was exclusively dependent on PPARβ/δ signaling, whereas the activation of TASK-1 was not. In addition, the PPARβ/δ-induced activation of K(Ca) was independent of NO. The acute prostacyclin-induced K(Ca) activation is critically dependent on PPARβ/δ as a rapid signaling factor. This accounts in part for the vasodilating effect of prostacyclin in pulmonary arteries, and provides insights into a new molecular explanation for the effects of prostanoids.

Thromboxane A(2) (TXA(2)) upregulates and activates NADPH oxidase (Nox) both of which are associated with cardiovascular disease. The aim of this study, therefore, was to investigate the relationship between thromboxane A(2) synthase (TXAS) status and Nox in human vascular smooth muscle cells (hVSMCs), in particular, whether superoxide (O(2)(▪-)) derived from Nox influences TXAS expression and activity. hVSMCs were incubated with TNFα: (10 ng/ml), TXA(2) mimetic U46619 (100 nM), 8-isoprostane F(2α) (8-IP; 100 nM) and hypoxia. Expression of TXAS was assessed using western blotting and quantitative PCR. The role of Nox1 and Nox4 was studied using apocynin and mRNA silencing. The effect of the thromboxane receptor antagonist picotamide and of iloprost, a prostacyclin (PGI(2)) analogue was also studied. TNF-α, U46619 and 8-IP and hypoxia all augmented TXAS expression as well as TXA(2) formation, effects inhibited by apocynin. Nox-1 (but not Nox4) gene silencing inhibited the increase in TXAS expression and activity. Both picotamide and iloprost inhibited the upregulation of TXAS as well as TXA(2) formation induced by TNF-α, U46619 and 8-isoprostane F(2α) and hypoxia. It is concluded that upregulation of TXA(2) synthase expression and activity in human VSMCs is mediated by an a priori upregulation of Nox1 and represents a self amplifying cascade. The inhibition of this effect with iloprost consolidates that PGI(2) plays a protective anti-oxidative role in the vasculature and that picotamide and like drugs may be effective in reducing the incidence of cardiovascular disease associated with an oxidative aetiology., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Rationale: Prostacyclin analogs, used to treat idiopathic pulmonary arterial hypertension (IPAH), are assumed to work through prostacyclin (IP) receptors linked to cyclic AMP (cAMP) generation, although the potential to signal through peroxisome proliferator-activated receptor-γ (PPARγ) exists., Objectives: IP receptor and PPARγ expression may be depressed in IPAH. We wished to determine if pathways remain functional and if analogs continue to inhibit smooth muscle proliferation., Methods: We used Western blotting to determine IP receptor expression in peripheral pulmonary arterial smooth muscle cells (PASMCs) from normal and IPAH lungs and immunohistochemistry to evaluate IP receptor and PPARγ expression in distal arteries., Measurements and Main Results: Cell proliferation and cAMP assays assessed analog responses in human and mouse PASMCs and HEK-293 cells. Proliferative rates of IPAH cells were greater than normal human PASMCs. IP receptor protein levels were lower in PASMCs from patients with IPAH, but treprostinil reduced replication and treprostinil-induced cAMP elevation appeared normal. Responses to prostacyclin analogs were largely dependent on the IP receptor and cAMP in normal PASMCs, although in IP(-/-) receptor cells analogs inhibited growth in a cAMP-independent, PPARγ-dependent manner. In IPAH cells, antiproliferative responses to analogs were insensitive to IP receptor or adenylyl cyclase antagonists but were potentiated by a PPARγ agonist and inhibited (∼ 60%) by the PPARγ antagonist GW9662. This coincided with increased PPARγ expression in the medial layer of acinar arteries., Conclusions: The antiproliferative effects of prostacyclin analogs are preserved in IPAH despite IP receptor down-regulation and abnormal coupling. PPARγ may represent a previously unrecognized pathway by which these agents inhibit smooth muscle proliferation.

Rationale: Mutations in the bone morphogenetic protein type II receptor (BMPR-II) are responsible for the majority of cases of heritable pulmonary arterial hypertension (PAH). Mutations lead to reduced Smad1/5-driven expression of inhibitor of DNA binding protein 1 (Id1) and loss of the growth suppressive effects of BMPs. The impact of existing PAH therapies on BMP signaling is lacking., Objective: Because prostacyclin analogues are effective treatments for clinical PAH, we hypothesized that these agents enhance Smad1/Id1 signaling., Methods and Results: Iloprost alone induced Id1 expression in human pulmonary artery smooth muscle cells (PASMCs), an effect that was independent of Smad1/5 activation but dependent on a cAMP-responsive element in the Id1 promoter. In addition, iloprost and treprostinil enhanced BMP-induced phosphorylation of Smad1/5 and Id1 expression in a cAMP-dependent manner. The mechanism involved suppression of inhibitory Smad, Smad6. Furthermore, iloprost rescued the deficit in Smad1/5 phosphorylation and Id gene expression in PASMCs harboring mutations in BMPR-II and restored growth suppression to BMP4 in mutant PASMCs. We confirmed a critical role for Id1 in PASMC proliferation. Reduced expression of Id1 was observed in concentric intimal lesions of heritable PAH cases. In the monocrotaline rat model of PAH, associated with reduced BMPR-II expression, we confirmed that treprostinil inhibited smooth muscle cell proliferation and prevented progression of PAH while enhancing Smad1/5 phosphorylation and Id1 gene expression., Conclusions: Prostacyclin analogues enhance Id1 expression in vitro and in vivo and restore deficient BMP signaling in BMPR-II mutant PASMCs.

The important athero-protective role of prostacyclin is becoming increasingly evident as recent studies have revealed adverse cardiovascular effects in mice lacking the prostacyclin receptor, in patients taking selective COX-2 inhibitors, and in patients in the presence of a dysfunctional prostacyclin receptor genetic variant. We have recently reported that this protective mechanism includes the promotion of a quiescent differentiated phenotype in human vascular smooth muscle cells (VSMC). Herein, we address the intriguing question of how localized endothelial release of the very unstable eicosanoid, prostacyclin, exerts a profound effect on the vascular media, often 30 cell layers thick. We report a novel PKA-, Akt-1- and ERK1/2-dependent prostacyclin-induced prostacyclin release that appears to play an important role in propagation of the quiescent, differentiated phenotype through adjacent arterial smooth muscle cells in the vascular media. Treating VSMC with the prostacyclin analog iloprost induced differentiation (contractile protein expression and contractile morphology), and also up-regulated COX-2 expression, leading to prostacyclin release by VSMC. This paracrine prostacyclin release, in turn, promoted differentiation and COX-2 induction in neighboring VSMC that were not exposed to iloprost. Using siRNA and pharmacologic inhibitors, we report that this positive feedback mechanism, prostacyclin-induced prostacyclin release, is mediated by cAMP/PKA signaling, ERK1/2 activation, and a novel prostacyclin receptor signaling pathway, inhibition of Akt-1. Furthermore, these pathways appear to be regulated by the prostacyclin receptor independently of one another. We conclude that prevention of de-differentiation and proliferation through a paracrine positive feedback mechanism is a major cardioprotective function of prostacyclin.

Pulmonary arterial hypertension (PAH) is a significant disease process characterized by elevated pulmonary vascular resistance leading to increased right ventricular afterload and ultimately progressing to right ventricular dysfunction and often death. Irreversible remodeling of the pulmonary vasculature is the hallmark of pulmonary hypertension and frequently leads to progressive functional decline in patients with PAH despite treatment with currently available therapies. Metabolites of the arachidonic acid cascade play an important homeostatic role in the pulmonary vasculature, and dysregulation of pathways downstream of arachidonic acid plays a central role in the pathobiology of PAH. Cyclooxygenase-2 (COX-2) is up-regulated in pulmonary artery smooth muscle cells (PASMC) and inflammatory cells during hypoxia and plays a protective role in the lung's response to hypoxia. We recently demonstrated that absence of COX-2 was detrimental in a mouse model of hypoxia-induced pulmonary hypertension. Exposure of COX-2 null mice to hypoxia resulted in severe pulmonary hypertension characterized by enhanced pulmonary vascular remodeling and significant up-regulation of the endothelin-1 receptor ET(A)R in the lung after hypoxia. Absence of COX-2 in vitro led to enhanced contractility of PASMC after exposure to hypoxia, which could be attenuated by iloprost, a prostaglandin I(2) analog. These findings suggest that selective inhibition of COX-2 may have detrimental pulmonary vascular consequences in patients with preexisting pulmonary hypertension or underlying hypoxemic lung diseases. Here, we discuss our recent data demonstrating the adverse consequences of COX-2 inhibition on pulmonary vascular remodeling and PASMC contractility.

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily that is highly expressed in enterohepatic tissue, is implicated in bile acid, lipid, and glucose metabolisms. Although recent studies showed that FXR is also expressed in vascular endothelial cells and smooth muscle cells, its physiological and/or pathological roles in vasculature tissue remain unknown. The aim of this study is to examine the chronic effect of synthetic FXR agonist GW4064 on vascular contraction and endothelium-dependent relaxation using tissue culture procedure. In cultured rabbit mesenteric arteries, the treatment with 0.1-10 microM GW4064 for 7 days did not influence vascular contractility induced by high K(+) (15-65 mM), norepinephrine (0.1-100 microM), and endothelin-1 (0.1-100 nM). However, the chronic treatment with GW4064 (1-10 microM for 7 days) dose dependently impaired endothelium-dependent relaxation induced by substance P (0.1-30 nM). In hematoxylin-eosin cross sectioning and en face immunostaining, GW4064 had no effects on the morphology of endothelial and smooth muscle cells. In endothelium-denuded arteries treated with GW4064 (1-10 microM) for 7 days, 3 nM-100 microM sodium nitroprusside-induced vasorelaxation, but not membrane-permeable cGMP analog 8-bromoguanosine-cGMP (8-Br-cGMP; 1-100 microM)-induced vasorelaxation, was significantly impaired. In these GW4064-treated arteries, 1 muM sodium nitroprusside-induced intracellular cGMP elevations were impaired. In RT-PCR, any changes were detected in mRNA expression level of alpha(1)- and beta(1)-subunit of soluble guanylyl cyclase. These results suggest that chronic stimulation of FXR impairs endothelium-dependent relaxation, which is due to decreased sensitivity of smooth muscle cells to nitric oxide.

Background and Purpose: To determine whether there is an association between vascular NADPH oxidase (NOX), superoxide, the small GTPase Rac(1) and PDE type 5 (PDE5) in human vascular smooth muscle cell (hVSMCs)., Experimental Approach: hVSMCs were incubated with xanthine-xanthine oxidase (X-XO; a superoxide generating system) or the thromboxane A(2) analogue, U46619 (+/-superoxide dismutase (SOD) or apocynin) for 16 h. The expression of PDE5 and NOX-1 was assessed using Western blotting and superoxide measured. The role of Rac(1) in superoxide generation was assessed by overexpressing either the dominant-negative or constitutively active Rac isoforms. The effects of iloprost, DETA-NONOate and the Rho-kinase inhibitor, Y27632, on PDE5 and NOX-1 expression were also studied., Key Results: Following 16 h incubation, U46619 and X-XO promoted the expression of PDE5 and NOX-1, an effect blocked by SOD or apocynin when co-incubated over the same time course. X-XO and U46619 both promoted the formation of superoxide. Overexpression of dominant-negative Rac(1) or addition of iloprost, DETA-NONOate or Y27632 completely blocked both superoxide release and PDE5 protein expression and activity., Conclusions and Implications: These data demonstrate that superoxide derived from NOX upregulates the expression of PDE5 in human VSMCs. As PDE5 hydrolyses cyclic GMP, this effect may blunt the vasculoprotective actions of NO.

Background: Cyclooxygenase-2 (COX-2) is upregulated in pulmonary artery smooth muscle cells (PASMCs) during hypoxia and may play a protective role in the response of the lung to hypoxia. Selective COX-2 inhibition may have detrimental pulmonary vascular consequences during hypoxia., Methods and Results: To investigate the role of COX-2 in the pulmonary vascular response to hypoxia, we subjected wild-type and COX-2-deficient mice to a model of chronic normobaric hypoxia. COX-2-null mice developed severe pulmonary hypertension with exaggerated elevation of right ventricular systolic pressure, significant right ventricular hypertrophy, and striking vascular remodeling after hypoxia. Pulmonary vascular remodeling in COX-2-deficient mice was characterized by PASMC hypertrophy but not increased proliferation. Furthermore, COX-2-deficient mice had significant upregulation of the endothelin-1 receptor (ET(A)) in the lung after hypoxia. Similarly, selective pharmacological inhibition of COX-2 in wild-type mice exacerbated hypoxia-induced pulmonary hypertension and resulted in PASMC hypertrophy and increased ET(A) receptor expression in pulmonary arterioles. The absence of COX-2 in vascular smooth muscle cells during hypoxia in vitro augmented traction forces and enhanced contractility of an extracellular matrix. Treatment of COX-2-deficient PASMCs with iloprost, a prostaglandin I(2) analog, and prostaglandin E(2) abrogated the potent contractile response to hypoxia and restored the wild-type phenotype., Conclusions: Our findings reveal that hypoxia-induced pulmonary hypertension and vascular remodeling are exacerbated in the absence of COX-2 with enhanced ET(A) receptor expression and increased PASMC hypertrophy. COX-2-deficient PASMCs have a maladaptive response to hypoxia manifested by exaggerated contractility, which may be rescued by either COX-2-derived prostaglandin I(2) or prostaglandin E(2).

Background: The over-production of superoxide (O(2)(-)) derived from NADPH oxidase (NOX) plays a central role in cardiovascular diseases. By contrast, nitric oxide (NO) and prostacyclin (PGI(2)) are vasculoprotective. The effect of the NO donor, NONOate and iloprost on O(2)(-) formation, p47(phox) and Rac(1) activation in human vascular smooth muscle cells (hVSMCs) was investigated., Methods: hVSMCs were incubated with 10nM thromboxane A(2) analogue, U46619 for 16h, and then with apocynin (a NOX inhibitor), NONOate or iloprost for 1h and O(2)(-) measured spectrophometrically. The role of cyclic AMP and cyclic GMP was examined by co-incubation of drugs with protein kinase (PK) A and G inhibitors listed above. Rac(1) was studied using pull-down assays., Results: NONOate and iloprost inhibited O(2)(-) formation, acutely, effects blocked by inhibition of PKG and PKA, respectively. Rac(1) and p47(phox) activation and translocation to the plasma membrane was completely inhibited by NONOate and iloprost, effects again reversed by co-incubation with PKG or PKA inhibitors., Conclusions: NO and PGI(2) block the acute activity of NOX in hVSMCs via the cGMP-PKG axis (for NO) and by the cAMP-PKA axis (for iloprost) through inhibition of Rac(1) and p47(phox) translocation. These findings have implications in the pathophysiology and treatment of CVD.

Objective: Stimulation of protease-activated receptor-1 (PAR-1) by thrombin causes vascular smooth muscle cell (SMC) mitogenesis and has been implicated in the vascular response to injury. Vascular injury is also associated with enhanced formation of PGE2 and PGI2 (prostacyclin). This study investigates whether PGI2 and PGE2 modify the expression of PAR-1 and the cellular response to thrombin in human SMC., Methods and Results: The PGI2-mimetic iloprost (1 to 100 nmol/L) attenuated mRNA, total protein, and cell surface expression of PAR-1. This was associated with inhibition of thrombin-induced mitogenesis and migration. Comparable inhibition of PAR-1 expression was observed with the selective IP-receptor agonist cicaprost, the adenylyl cyclase activator forskolin, the phosphodiesterase inhibitor isobutylmethylxanthine and the PKA activator dibutyryl-cAMP. Similar effects of PGE2 required micromolar concentrations. The specific PKA-inhibitor Myr-PKI prevented PAR-1 downregulation by iloprost. The potential role of Rho family GTPases in PAR-1 regulation was also investigated. Iloprost decreased Rac1 mRNA and the Rac1 inhibitor NSC23766 mimicked the inhibitory effects of iloprost on PAR-1 protein--but not mRNA. The Rho kinase inhibitor Y27632 did not influence PAR-1 expression., Conclusions: IP-receptor agonists may limit the mitogenic actions of thrombin in human SMC by downregulating PAR-1 via modulation of cAMP-/PKA- and Rac1-dependent signaling pathways.

We have previously shown that interleukin (IL)-1beta, transforming growth factor (TGF)-beta1, or bradykinin (BK) impair cAMP generation in response to prostacyclin analogs in human pulmonary artery smooth muscle (PASM), suggesting that inflammation can impair the effects of prostacyclin analogs on PASM in pulmonary hypertension. Here we explored the biochemical mechanisms involved. We found that IL-1beta, BK, and TGF-beta1 reduced adenylyl cyclase isoform 1, 2, and 4 mRNA, increased Galphai protein levels, and reduced prostacyclin receptor (IP receptor) mRNA expression. In contrast, Galphas protein levels were unchanged. Protein kinase A (PKA) (H-89, KT-2750, PKIm) and p38 mitogen-activated protein (MAP) kinase (SB-202190) inhibitors attenuated these effects, but protein kinase C (bisindolylmaleide) or phosphoinositol 3-kinase (LY-294002) inhibitors did not. Fluorescent kemptide assay and Western blotting confirmed that PKA and p38 MAP kinase were activated by IL-1beta, BK, and TGF-beta1. These studies suggest that IL-1beta, BK, and TGF-beta1 impair IP receptor-mediated cAMP accumulation by multiple effects on different components of the signaling pathway and that these effects are PKA and p38 MAP kinase dependent.

Background: Prostacyclin (PGI2) and thromboxane (TxA2) effect disparate outcomes for atherogenesis and the response to vascular injury; PGI2, a vasodilator and inhibitor of platelet aggregation, limits the deleterious actions of TxA2, a vasoconstrictor and platelet activator. Dimerization of their G protein-coupled receptors, IP and TP, evokes a modified cellular response through which IP/TP counter-balance may be effected. We examined the consequence of IP/TP interaction for the regulatory pathways of both receptors., Methods and Results: TPalpha overexpressed in HEK293 cells or expressed endogenously in aortic smooth muscle cells (ASMCs) was internalized after selective activation of either TP or IP. Homologous trafficking of TP was unaltered by coexpression of IP. Heterologous sequestration of TPalpha required the physical presence of activated IP, in transfected and native cells, but was independent of IP signaling to adenylyl cyclase. Reciprocal heterologous regulation of IP, via activated TP, was evident in both HEK293 cells and ASMCs. Homologous TP internalization led to receptor retention and degradation. In contrast, when internalization was IP-induced, TPalpha was recycled to the cell surface in coexpressing HEK293 cells, but not in ASMCs, in accord with the postendocytotic pathway of IP., Conclusions: IP/TPalpha interaction permits reciprocal regulation of receptor endocytosis via the trafficking pathway determined by the activated dimeric partner.

Background: Pulmonary arterial hypertension is a proliferative vascular disease, characterized by aberrant regulation of smooth muscle cell proliferation and apoptosis in distal pulmonary arteries. Prostacyclin (PGI2) analogues have anti-proliferative effects on distal human pulmonary artery smooth muscle cells (PASMCs), which are dependent on intracellular cAMP stimulation. We therefore sought to investigate the involvement of the main cAMP-specific enzymes, phosphodiesterase type 4 (PDE4), responsible for cAMP hydrolysis., Methods: Distal human PASMCs were derived from pulmonary arteries by explant culture (n = 14, passage 3-12). Responses to platelet-derived growth factor-BB (5-10 ng/ml), serum, PGI2 analogues (cicaprost, iloprost) and PDE4 inhibitors (roflumilast, rolipram, cilomilast) were determined by measuring cAMP phosphodiesterase activity, intracellular cAMP levels, DNA synthesis, apoptosis (as measured by DNA fragmentation and nuclear condensation) and matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) production., Results: Expression of all four PDE4A-D genes was detected in PASMC isolates. PDE4 contributed to the main proportion (35.9 +/- 2.3%, n = 5) of cAMP-specific hydrolytic activity demonstrated in PASMCs, compared to PDE3 (21.5 +/- 2.5%), PDE2 (15.8 +/- 3.4%) or PDE1 activity (14.5 +/- 4.2%). Intracellular cAMP levels were increased by PGI2 analogues and further elevated in cells co-treated with roflumilast, rolipram and cilomilast. DNA synthesis was attenuated by 1 microM roflumilast (49 +/- 6% inhibition), rolipram (37 +/- 6%) and cilomilast (30 +/- 4%) and, in the presence of 5 nM cicaprost, these compounds exhibited EC50 values of 4.4 (2.6-6.1) nM (Mean and 95% confidence interval), 59 (36-83) nM and 97 (66-130) nM respectively. Roflumilast attenuated cell proliferation and gelatinase (MMP-2 and MMP-9) production and promoted the anti-proliferative effects of PGI2 analogues. The cAMP activators iloprost and forskolin also induced apoptosis, whereas roflumilast had no significant effect., Conclusion: PDE4 enzymes are expressed in distal human PASMCs and the effects of cAMP-stimulating agents on DNA synthesis, proliferation and MMP production is dependent, at least in part, on PDE4 activity. PDE4 inhibition may provide greater control of cAMP-mediated anti-proliferative effects in human PASMCs and therefore could prove useful as an additional therapy for pulmonary arterial hypertension.

Prostaglandin E(2) produced endothelium-independent relaxation of phenylephrine- and 5-HT-contracted piglet saphenous vein (PSV; pEC(50)=8.6+/-0.2; n=6). The prostanoid EP(4) receptor antagonist GW627368X (30-300 nM) produced parallel rightward displacement of PGE(2) concentration-effect (E/[A]) curves (pK(b)=9.2+/-0.2; slope=1). Higher concentrations of GW627368X did not produce further rightward shifts, revealing the presence of non-EP(4) prostanoid receptors. In all, 18 other prostanoid receptor agonists relaxed PSV in a concentration-related manner. Relative potencies of agonists most sensitive to 10 muM GW627368X (and therefore predominantly activating EP(4) receptors) correlated well with those at human recombinant EP(4) receptors in human embryonic kidney (HEK-293) cells (r(2)=0.74). In the presence of 10 microM GW627368X, the rank order of agonist relative potency matched that of the human recombinant EP(2) receptor in Chinese hamster ovary cells (r(2)=0.72). Iloprost, cicaprost and PGI(2) relaxed PSV maximally and were antagonised by 10 microM GW627368X, demonstrating that they were full EP(4) receptor agonists. Residual responses to these compounds in the presence of GW627368X suggested the presence of IP receptors.BW245C relaxed PSV maximally (pEC(50)=6.8+/-0.1). In the presence of 10 microM GW627368X, BW245C produced biphasic E/[A] curves (phase one pEC(50)=6.6; alpha=24%; phase two pEC(50)=5.1; alpha=112%). Phase two was antagonised by the DP receptor antagonist BW A868C (1 microM), demonstrating that BW245C is an agonist at DP and EP4 receptors. We conclude that PSV contains EP(4), EP(2), DP and IP receptors; IP receptor agonists are also porcine EP(4) receptor agonists.

Recent studies have demonstrated that cerebral arteries from rats fed a high-salt (HS) diet exhibit impaired vasodilation and altered electrophysiological response to reduction in PO2. The present study examined whether an increase in salt intake alters the response of vascular smooth muscle cells (VSMC) to prostacyclin, a crucial mediator of hypoxic dilation in cerebral arteries. VSMC were isolated from cerebral arteries of male Sprague-Dawley rats maintained on an HS (4% NaCl) or a low-salt diet (0.4% NaCl) for 3 days. The stable prostacyclin analog iloprost (10 ng/ml) inhibited serotonin (0.1-10 microM)-induced contractions and the increase in intracellular Ca2+ concentration ([Ca2+]i) in VSMC isolated from arteries of animals fed the low-salt diet. In contrast, iloprost had no effect on serotonin-induced contractions and increases in [Ca2+]i in VSMC isolated from arteries of rats fed the HS diet. Preventing the fall in ANG in rats fed the HS diet by infusion of a low dose of ANG II (5 ng.kg(-1).min(-1) i.v.) restored the inhibitory effect of iloprost on serotonin-induced contractions and increases in [Ca2+]i in VSMC from animals fed the HS diet. These effects were reversed by AT1 receptor blockade with losartan. These results indicate that ANG II suppression secondary to elevated dietary salt intake impairs vascular relaxation and Ca2+ regulation by prostacyclin.

Prostanoids can suppress vascular smooth muscle cell (VSMC) proliferation, but the mechanism through which this is mediated has not been identified. In this study, we show rat aortic VSMCs to express the EP1, EP2, EP3, EP4, and IP receptors. The EP4 receptor-specific agonist, 11-deoxy-PGE1, induced a time-dependent phosphorylation of protein kinase C and extracellular signal-regulated kinase (ERK) 1/2 in serum-depleted (0.1%) VSMCs, whereas the EP2 receptor agonist, butaprost, was without effect. PGI2 or iloprost at the IP receptor inhibited basal ERK phosphorylation with IC50 values of 10 nmol/L. Iloprost also attenuated the sustained activation of ERK induced by endothelin-1 or basic fibroblast growth factor (bFGF). Endothelin-1 or bFGF significantly increased the number of VSMCs counted 24 hours later compared with basal, and both responses were blocked by the MEK inhibitor, U0126, or iloprost. Under basal conditions, U0126 or iloprost reduced the number of viable cells and increased caspase-3 activity, which could be reversed by coapplication with endothelin-1, bFGF, or the adenylate cyclase inhibitor, SQ22536. Endothelin-1, bFGF, or SQ22536 prevented the depression to below basal levels of ERK phosphorylation induced by iloprost. Forskolin activated caspase-3 and attenuated basal ERK phosphorylation, which were prevented by SQ22536, endothelin-1, or bFGF. These data suggest that iloprost induces apoptosis via a cAMP-mediated suppression of ERK activity. In turn, this apoptotic response can be blocked by a mitogenic stimulus that re-establishes ERK activity back to basal levels, but at the expense of any concomitant proliferative activity. However, ERK stimulation by a selective EP4 receptor agonist, suggests that prostanoids may have diverse and complex roles in VSMC physiology.

Hyaluronic acid (HA) is a prominent constituent of the extracellular matrix of atherosclerotic vascular lesions in humans known to modulate vascular smooth muscle phenotype. The regulation of HA synthesis by vasodilatory prostaglandins was analyzed in human arterial smooth muscle cells (SMCs). The prostacyclin analogue, iloprost (100 nmol/L), markedly increased pericellular formation of HA coats and HA secretion into the cell culture medium in human arterial SMCs (8.7+/-1.6-fold). Expression of HA synthase 2 (HAS2) was determined by semiquantitative RT-PCR and found to be strongly upregulated at concentrations of iloprost between 1 and 100 nmol/L after 3 hours. Furthermore, endogenous cyclooxygenase-2 (COX2) activity was required for basal expression of HAS2 mRNA in SMCs in vitro. Total HA secretion in response to iloprost was markedly decreased by RNA interference (RNAi), specific for HAS2. In addition, siRNA targeting HAS2 strongly increased the spreading of human SMCs compared with mock-transfected cells. HAS2 mRNA levels were also stimulated by a selective prostacyclin receptor (IP) agonist, cicaprost (10 nmol/L), prostaglandin E(2) (10 nmol/L), and the EP(2) receptor agonist, butaprost (1 micromol/L). Induction of HAS2 mRNA and HA synthesis by prostaglandins was mimicked by stable cAMP analogues and forskolin. In human atherectomy specimens from the internal carotid artery, HA deposits and COX2 expression colocalized frequently. In addition, strong EP(2) receptor expression was detected in SMCs in HA-rich areas. Therefore, upregulation of HAS2 expression via EP(2) and IP receptors might contribute to the accumulation of HA during human atherosclerosis, thereby mediating proatherosclerotic functions of COX2.

Migration and proliferation of medial smooth muscle cells (SMC) in the arterial intima contributes to the development of atherosclerotic plaques and restenotic processes after coronary angioplasty. Prostacyclin (PGI2)-mediated stimulation of cyclic adenosine 3'5'-monophosphate (cAMP) signaling is believed to be important for maintaining SMC in a quiescent state. In order to identify new cellular targets of PGI2/cAMP action, we have used microarray screening to examine changes in the transcriptional profile in human vascular SMC in response to exposure to the stable PGI2 mimetic iloprost. We have identified 83 genes with significantly altered expression after iloprost (100 nM) exposure for 6 hr. Fifty-one genes were upregulated, among them stanniocalcin precursor (18.8+/-2.7), zinc finger transcription factor (7.8+/-2.0), hyaluronan synthase 2 (6.8+/-1.8), cyclooxygenase 2 (4.7+/-0.8), dual specific phosphatase (3.9+/-0.5) and vascular endothelial growth factor (2.3+/-0.4). Thirty-two genes were reduced, among them cystein-rich angiogenic protein (-14.9+/-1.3), monocyte chemotactic protein 1 (-7.4+/-1.1) and plasminogen activator inhibitor PAI-1 (-4.5+/-0.5). By means of semi-quantitative RT-PCR, time-courses of gene expression were established. The present study identified genes not hitherto recognized to be targets of PGI2 action, providing further insight into its cAMP-mediated effects on SMC growth, migration and matrix secretion.

Prostacyclin and its mimetics have repeatedly been shown to act antiatherogenic and to inhibit neointima formation in several animal models of vascular injury. Treatment of human vascular smooth muscle cells with the prostacyclin mimetic iloprost (100 nm) drastically reduces expression of Cyr61, encoding the growth-regulatory cystein-rich angiogenic protein, without affecting the degradation rate of Cyr61 mRNA. Thrombin-induced Cyr61 expression was inhibited completely in the presence of iloprost. It is concluded that vasoprotective actions of prostacyclin in vivo may in part be due to inhibition of expression of the growth regulatory gene Cyr61 at sites of vascular lesions.

Prostaglandin-endoperoxide synthase-2 (PGH-synthase) or cyclooxygenase-2 (COX-2) is inducible by a variety of stimuli, e.g. inflammatory mediators, growth factors and hormones and is believed to be responsible for the majority of inflammatory prostanoid production. Moreover, it has been demonstrated that COX-2 contributes substantially to prostacyclin-synthesis in patients with atherosclerosis. In this study, we demonstrate an up-regulation of COX-2 mRNA, protein and product formation by the prostacyclin-mimetic iloprost in human vascular smooth muscle cells (hSMC). COX-2 mRNA expression was induced transiently between 1 and 6 hr and returned to basal levels after 16 hr of iloprost stimulation. COX-2 protein was induced concomitantly between 3 and 6 hr of iloprost stimulation. This was accompanied by an increase in PGI(2) formation. Forskolin, a direct activator of adenylyl cyclase, and dibutyryl cAMP, a cell-permeable cAMP analogue-induced COX-2 mRNA, suggesting a cAMP-dependent COX-2 expression in hSMC. Iloprost-induced COX-2 protein expression and PGI(2) formation was synergistically elevated by co-stimulation with the phorbolester PMA (phorbol-12-myristate-13-acetate). It is concluded, that the observed up-regulation of COX-2 with subsequent release of newly synthesized PGI(2) and the synergistic effect of iloprost and phorbolester on PGI(2) formation provide a positive feedback of prostaglandins on their own synthesizing enzyme. This might be important for control of hSMC proliferation, migration and differentiation as well as inhibition of platelet aggregation.

Primary pulmonary hypertension is characterized by increased pulmonary vascular resistance and smooth muscle proliferation. Stable analogs are increasingly being used to treat this disease, although no data exists comparing their effects on proliferation. We therefore investigated the antiproliferative activity of several prostacyclin (PGI(2)) analogs on human pulmonary arterial smooth muscle cells, including UT-15 and iloprost, analogs that have recently completed successful clinical trials. Serum-induced proliferation, as assessed by [(3)H]thymidine incorporation (30 h) or cell number (48 h), was significantly inhibited with a 10-fold difference in potency, ranking in effectiveness UT-15 > iloprost > cicaprost > beraprost. Effects were reversed by the adenylyl cyclase inhibitor, 2,5'dideoxyadenosine (DDA) but not SQ22536. Intracellular cyclic AMP (cAMP) was elevated by all analogs and inhibited by DDA, although SQ22536 was a highly variable inhibitor, suggesting that different pathways might mediate cAMP generation. UT-15 produced a significantly larger and more sustained increase in cAMP compared with other analogs, with iloprost being the weakest elevator. Thus, PGI(2) analogs potently inhibit proliferation of human pulmonary artery, probably via a cAMP-dependent pathway, although cAMP elevation in itself is not a good predictor of antiproliferative potency.

Diabetes is associated with gender-specific macrovascular complications arising from increased oxidant stress in the vascular wall. In this study, male and female rats were treated with two structurally unrelated drugs sharing antioxidant properties, lercanidipine and Leucoselect (both 3 mg/kg/day), for 1 week starting 1 day after streptozotocin-diabetes induction. Concentration-response curves to L-nitroarginine methylester (L-NAME), superoxide dismutase and acetylcholine in aortic rings showed significantly greater nitric oxide-mediated relaxation in female compared with male non-diabetic rats. Diabetes increased contractility to noradrenaline and L-NAME in both genders, whereas relaxation to acetylcholine and iloprost were significantly attenuated in females only. Treatment with lercanidipine and Leucoselect restored, at least in part, responses to noradrenaline, acetylcholine and iloprost without affecting those to L-NAME and sodium nitroprusside. Unexpectedly, both drugs impaired superoxide dismutase response in female tissues. In conclusion, female rat aorta is markedly exposed to short-term diabetic vascular injury, which may be prevented by antioxidant treatment.

Prostacyclin permissively allows increased cAMP and cerebral vasodilation to hypercapnia in piglets. The prostacyclin receptor (IP) is coupled to phospholipase C (PLC) in piglet cerebral microvascular smooth muscle cells (SMC). We hypothesize that inhibition of PLC blocks the permissive action of IP receptor agonist, iloprost, and direct activation of PKC substitutes for the IP receptor agonist in SMC. SMC cAMP production was measured at normal pHi/pHo and with reduced pHi/pHo in the absence and presence of iloprost (100 pM). Half of the cells were pretreated with U73122, the PLC inhibitor, which decreased the basal IP3 and blocked the increase in IP3 caused by iloprost. Without iloprost, decreasing pHi/pHo increased cAMP production (40%). With iloprost, the cAMP response to acidosis increased to over 80%. U73122 prevented accentuation of the cAMP response by iloprost. Phorbol myristate acetate augmented the response to acidosis similarly to iloprost. These data suggest IP agonists augment the cAMP response to acidosis via coupling through PLC to activate PKC.

In a previous study of cultured preglomerular vascular smooth muscle cells, it was demonstrated that, although the stable prostacyclin analog iloprost alone had no effect on the intracellular calcium concentration ([Ca2+](i)), it did significantly attenuate the increase in [Ca2+](i) stimulated by angiotensin II (AngII). In this study, the mechanisms by which iloprost interacts with calcium signaling pathways stimulated by AngII were examined. [Ca2+](i) was assessed using the calcium-sensitive fluorescent dye fura-2. Initial studies identified two major components of the [Ca2+](i) response to AngII in this homogeneous preparation of vascular smooth muscle cells from renal resistance vessels. Mobilization of internal stores was evident as an immediate TMB-8-sensitive peak increase in [Ca2+](i) (52 +/- 6 to 297 +/- 26 nM, P: < 0.001) in a calcium-free medium. After [Ca2+](i) had returned to baseline levels during continued AngII stimulation, a nifedipine-sensitive entry pathway was revealed by the sustained stimulatory effect of added external calcium, which increased [Ca2+](i) to 112 +/- 13 nM (P: < 0.001). Coadministration of iloprost with AngII attenuated both the immediate peak (154 +/- 14 nM) and sustained plateau (61 +/- 9 nM) phases. Increases in endogenous levels of cAMP induced by the phosphodiesterase inhibitor milrinone mirrored the actions of iloprost, suggesting that the prostacyclin analog exerted its actions via cAMP activation. Blockade of cAMP-dependent protein kinase with KT 5720 reversed the effects of both iloprost and milrinone. When iloprost or milrinone was introduced after the initial mobilization peak had dissipated, the plateau phase of calcium entry was unchanged (92 +/- 9 nM). The concept that iloprost does not directly modulate calcium entry was further supported by data showing that the activation of L-type calcium channels by BAY-K 8644 was unchanged during iloprost treatment. On the basis of the observation that iloprost did not alter thapsigargin stimulation of Ca(2+)-ATPase activity, it is concluded that the actions of cAMP are distinct from increasing calcium uptake into the sarcoplasmic reticulum. This study provides new information on the ability of iloprost to primarily attenuate inositol-1,4,5-triphosphate-mediated calcium mobilization via cAMP, with secondary inhibition of L-type calcium entry channels. These data clarify the mechanism by which prostaglandins buffer AngII constriction in resistance arterioles.

Background: Prostacyclin has proved to be a beneficial treatment for patients with severe pulmonary hypertension. We postulated that the response may reflect, at least in part, inhibition of pulmonary artery smooth muscle cell (PASMC) growth., Methods and Results: Human PASMCs were derived from distal (<1-mm external diameter, n=8) and proximal (>8-mm external diameter, n=12) pulmonary arteries obtained at transplant surgery and pneumonectomy. The effects of the stable prostacyclin analogues on [methyl-(3)H]thymidine incorporation and cell proliferation were investigated by using immunohistochemically characterized cells. Distal cells proliferated faster than did proximal PASMCs and displayed a distinct sensitivity to cicaprost and iloprost. Both analogues inhibited thymidine uptake over 24 hours (20% to 60%, P<0.001; n=8) and abolished stimulation of DNA synthesis by platelet-derived growth factor-BB (10 ng/mL) in distal but not proximal cells. The inhibitory effect of cicaprost was mimicked by isoproterenol (10(-5) mol/L), forskolin (10(-5) mol/L), and dibutyryl cAMP (5x10(-4) mol/L) and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (5x10(-5) mol/L). Cicaprost (10(-10) to 10(-6) mol/L) inhibited the proliferation of PASMCs, which had been stimulated with either platelet-derived growth factor-BB or serum, and increased cAMP production. These effects were potentiated by 3-isobutyl-1-methylxanthine and attenuated by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (10(-5) to 10(-4) mol/L)., Conclusions: ++Cicaprost and iloprost inhibit DNA synthesis and proliferation to a greater extent in distal compared with proximal human PASMCs, acting at least in part via a cAMP-dependent mechanism. The results are consistent with the hypothesis that prostacyclin analogues inhibit vascular remodeling in pulmonary hypertension and demonstrate heterogeneity among human PASMCs.

The mechanism through which iloprost permits cerebral vasodilation induced by specific stimuli is incompletely understood. Previous study suggests there might be interplay between the adenylyl cyclase and phospholipase C (PLC) systems. Coupling of the prostacyclin receptor with the PLC pathway system was investigated. Iloprost, a stable prostacyclin analog, was used as a prostacyclin receptor agonist. We investigated the effects of iloprost (10-12-10-6 M) on inositol 1,4,5-trisphosphate (IP3) production by piglet cerebrovascular smooth muscle cells in primary culture. Iloprost caused concentration- and time-dependent increases in IP3 production in control cells and in cells pretreated with LiCl (to prevent further IP3 metabolism). Iloprost treatment (10-12 M) of cerebrovascular smooth muscle cells, in the absence and presence of 20 mM LiCl, resulted in 2-fold and 4-fold increases in the formation of IP3, respectively. In contrast, 10-10 M to 10-6 M iloprost, either in the presence or absence of LiCl, induced moderate or no increase in IP3 formation. Iloprost (10-10-10-12 M) strongly stimulated diacylglycerol (DAG) generation, whereas higher concentrations (10-8 M) did not induce an increase. In conclusion, the results suggest that prostacyclin receptors on cerebromicrovascular smooth muscle can couple to PLC, generating the second messengers, IP3 and DAG.

In order to exert an appropriate biological effect, the action of the vasoconstrictive hormone angiotensin II (ANG II) is modulated by vasoactive factors such as prostaglandins PGE2 and PGI2. The present study investigates whether prostaglandins alter ANG II-mediated increases in cytosolic calcium concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC) isolated from rat renal preglomerular arterioles. [Ca2+]i was assessed using the calcium-sensitive dye fura 2 and a microscope-based photometer system. ANG II (10(-7) M) caused a biphasic, time-dependent [Ca2+]i response: an initial peak increase from 52 +/- 7 to 264 +/- 25 nM, followed by a sustained plateau of 95 +/- 9 nM in cultured VSMC. Coadministration of PGE2 or PGI2 or synthetic mimetics caused dose-dependent decreases in the peak [Ca2+]i response to ANG II, with attenuation of 40-50%. This degree of inhibition was even more pronounced in individual freshly isolated preglomerular VSMC. Increasing cAMP levels in cultured VSMC, by using either a cell-permeable analog or inhibiting phosphodiesterase activity, mirrored the antagonistic effects of prostaglandins on ANG II-stimulated increases in [Ca2+]i. Radioimmunoassays demonstrate that ANG II (10(-7) M) stimulates production of PGI2 and PGE2; the stable prostacyclin metabolite 6-keto-PGF(1alpha) was released in 10-fold greater concentrations than PGE(2.) Indomethacin blockade of prostaglandin production potentiated both the peak (264 to 337 +/- 26 nM) and sustained [Ca2+]i responses (95 to 181 +/- 22 nM) to ANG II. When prostaglandin analogs were added during indomethacin treatment, the ANG II response was restored to the typical pattern. In conclusion, we demonstrate that modulation of intracellular calcium levels is one mechanism by which prostaglandins can buffer ANG II-mediated constriction in renal preglomerular VSMC. PGI2 is more potent than PGE2 in this regard.

In newborn pig pial arterioles and cocultures of cerebral microvascular endothelial and smooth muscle cells, hypercapnia increases cAMP. In the intact cerebral circulation, both the increase in cAMP and the accompanying vasodilation require the presence of PGI(2). Using piglet cerebral microvascular smooth muscle in primary culture, we addressed the hypothesis that, in the presence of PGI(2), hypercapnia-induced changes in extracellular pH cause increases in cAMP. The stable PGI(2)-receptor agonist iloprost did increase production of cAMP in response to combined extracellular pH and pH(i) (11 +/- 6 vs. 32 +/- 10% in the absence and presence of 10(-10) M iloprost, respectively). However, there was no positive dose-response relationship between iloprost concentration and stimulation of cAMP production by acidosis (e.g., 58 +/- 9 vs. 41 +/- 5% in the presence of 10(-12) and 10(-9) M iloprost, respectively). Rapid decreases in pH(i) stimulate the cAMP production. Decreases in extracellular pH do not appear to contribute further. The G protein inhibitor pertussis toxin did not augment cAMP production in response to decreasing pH(i). We conclude that PGI(2) receptor activation permits another mechanism to enhance cAMP generation in response to intracellular, but not extracellular, acidosis and that the mechanism of the permissive effect of PGI(2) does not involve inhibition of a pertussis toxin-sensitive G protein.

The role of K+ channels in mediating vasorelaxation induced by two prostacyclin analogues was investigated in guinea-pig aorta. Iloprost caused substantial relaxation of tissues contracted with phenylephrine or 25 mM K+ but not 60 mM K+. In endothelial-denuded tissues, maximal relaxations to iloprost, cicaprost or isoprenaline were inhibited by approximately 40-50% with tetraethylammonium or iberiotoxin, both blockers of large conductance Ca2+-activated K+ (BKCa) channels. In contrast, the response to forskolin, an activator of adenylate cyclase was marginally inhibited by tetraethylammonium. The K(ATP) channel blocker, glibenclamide significantly augmented the response to iloprost but not cicaprost. These effects were largely inhibited by the EP1 receptor antagonist, 8-chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid 2-[1-oxo-3(4-pyridinyl)propyl]hydrazide, monohydrochloride (SC-51089) and partially by indomethacin, suggesting that iloprost relaxation is counterbalanced by activation of EP1 receptors, in part through a constrictor prostaglandin. We conclude that BKCa channels play an important role in mediating the effects of iloprost and cicaprost and raises the possibility that cyclic AMP-independent pathways might be involved.

The present study of newborn pig cerebral circulation investigated the role of pertussis toxin (PTX)-sensitive GTP binding proteins in the permissive action of prostacyclin in specific dilator responses. Pial arterioles of anesthetized piglets were observed through closed cranial windows. The piglets were treated topically with PTX and intravenously with indomethacin. The effects of hypercapnia (10% CO2 ventilation) and topical 5,6-epoxyeicosatrienoic acid (5,6-EET) on pial arteriolar diameter were noted before and after the intervention. Samples of the artificial cerebrospinal fluid (aCSF) were collected from beneath the cranial windows for determination of the cAMP concentration. After administration of PTX, indomethacin still abolished pial arteriolar dilation to both hypercapnia and 5, 6-EET and also inhibited the cAMP elevation caused by hypercapnia. The addition of phorbol 12-myristate 13-acetate (PMA), but not iloprost, restored the increase in cAMP and vascular responses to hypercapnia and 5,6-EET. Therefore, in the newborn pig cerebral microvasculature, PTX appears to inhibit a G protein involved in the permissive action of prostacyclin. However, the protein kinase C (PKC) activator PMA appears to act downstream from the block, and, therefore, the permissive action of PMA is not affected by PTX. We suggest that the prostacyclin IP receptor may be coupled to phospholipase C via a PTX-sensitive G protein that normally permits vasodilation to specific stimuli via activation of a PKC, resulting in phosphorylation of a component of the adenylyl cyclase pathway.

Purpose: The authors have previously reported that intramural delivery of iloprost during angioplasty suppresses local platelet aggregation at 1 hour in undiseased porcine arteries. In this study, the authors sought to quantify the effect of such treatment on medial vascular smooth muscle cell proliferation, an event implicated in the development of intimal hyperplasia., Materials and Methods: Three Yorkshire pigs underwent percutaneous transluminal angioplasty with hydrogel-coated balloons for a total of 10 iloprost-treated (experimental) and 10 saline-treated (control) arterial sites. The balloons were prepared with previously reported techniques and loaded with 2.25 microg of iloprost for the experimental sites. On the eighth day after angioplasty, these sites were harvested and prepared for immunohistochemical staining. Thin (4 microm) sections of the specimens were stained with use of monoclonal antibody to proliferating cell nuclear antigen (PCNA). Appropriate positive and negative controls were used. Approximately 350-500 vascular smooth muscle cells were randomly counted under high power (100x) by an experienced physician who was blinded to the origin of the specimen. A PCNA index (%) was calculated as follows: [(#PCNA [+] cells)/(#PCNA [+] cells + #PCNA [-] cells)]x 100. A paired t test was used for statistical comparison., Results: The PCNA indices for eight (n = 8) paired large vessels (iliac, carotid, subclavian) were 7.98 (+/- 1.8)%, for the iloprost-treated experimental sites, and 14.58 (+/- 3.8)% for the saline-treated control sites. This difference was statistically significant (P = .003). One large vessel pair was not available for analysis. When the pair of renal arteries of animal 3 were included (n = 9), the PCNA indices were 8.32 (+/- 2.3)% for the experimental sites, and 13.79 (+/- 4.2)% for the control sites. The differences were again significant (P = .01)., Conclusion: Intraarterial site-specific delivery of iloprost during angioplasty with drug-loaded, hydrogel-coated balloons significantly suppresses medial smooth muscle cells in swine at the expected peak period of proliferation of 7 days after angioplasty.

This study compares the antimitogenic effects of iloprost and prostaglandin E1 on platelet-derived growth factor-BB stimulated DNA synthesis ([3H]thymidine incorporation) in bovine coronary artery smooth muscle cells. When added 20-24 h after stimulation with platelet-derived growth factor-BB (20 ng/ml), both iloprost and prostaglandin E1, concentration-dependently (IC50 3-5 nM) inhibited DNA synthesis. However, when added together with the growth factor (0-24 h), the inhibition of DNA synthesis by iloprost was markedly attenuated, indicating tolerance development. In contrast, no tolerance to antimitogenic effects of prostaglandin E1 or forskolin were observed. When added to iloprost-tolerant cells, both prostaglandin E1 and forskolin, still inhibited DNA synthesis. There was no evidence for transcriptional down-regulation of prostacyclin receptor gene by iloprost. The data demonstrate a tolerance development to antimitogenic actions of prostacyclin but not of prostaglandin E1 and suggest that the receptors, mediating the antiproliferative actions of these prostaglandins, may be different.

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10(-5) M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms., (Copyright 1998 Academic Press.)

Activation of a soluble guanylyl cyclase plays an important role in nitric oxide (NO)-induced vasodilation. Recently, we have reported that NO increases the calcium-activated potassium (K(Ca)) channel activity in vascular smooth muscle cells from coronary arteries. The present study examined the role of the soluble guanylyl cyclase in the control of basal activity of the K(Ca) channels and in mediating NO-induced activation of the K(Ca) channels in vascular smooth muscle cells, using a selective inhibitor of this enzyme, 1H-[1,2,4]oxadiazolo[4,2-alpha]quinoxalin-1-one (ODQ). In the cell-attached patch-clamp mode, addition of ODQ into the bath solution (10 micromol/L) decreased the K(Ca) channel activity by 59% and attenuated activation of the channels induced by the NO donor, deta nonoate, by 70%. ODQ had no effect on 8-bromo-cGMP-induced activation of the K(Ca) channels. Deta nonoate produced a concentration-dependent relaxation of precontracted coronary arteries. When ODQ was added to the bath, the deta nonoate-induced relaxations were inhibited. The IC50 for deta nonoate was decreased by about 25-fold and the maximal effect of deta nonoate was reduced by about 60%. A specific K(Ca) channel inhibitor, iberiotoxin, decreased deta nonoate-induced vasodilation but to a lesser extent than ODQ. However, ODQ was without effect on the vasodilation induced by a prostacyclin analog, iloprost, and by adenosine. These results indicate that a soluble guanylyl cyclase and cGMP play an important role in the control of the K(Ca) channel activity in coronary arterial smooth muscle cells. K(Ca) channel activation participates in the NO-induced vasodilation in coronary circulation.

The specific prostacyclin (IP) receptor agonist cicaprost relaxed human pulmonary artery preparations precontracted with phenylephrine [50% inhibitory concentration (IC50) approximately 0.6 nM], U-46619 (IC50 approximately 0.9 nM), and K+ (approximately 40% maximal relaxation); endothelium removal had little effect on relaxant activity. Ranking of relaxant potencies for prostacyclin and five of its analogs was 17 alpha, 20-dimethyl-delta 6,6a-6a-carba PGI1 (TEI-9063) > or = cicaprost > iloprost > prostacyclin > taprostene > benzodioxane prostacyclin > 15-deoxy-16 alpha-hydroxy-16 beta,20-dimethyl-delta 6,6a-6a-carba PGI1 (TEI-3356). The potency of the isocarbacyclin TEI-3356 may have been under-estimated because of its contractile (EP3 receptor agonist) activity. The potency ranking of four nonprostanoid prostacyclin mimetics was 3-[4-(4,5-diphenyl-2-oxazolyl)-5-oxazolyl]phenoxy] acetic acid (BMY 45778; IC50 approximately 2.5 nM) > > 2-[3-[2-(4, 5-diphenyl-2-oxazolyl)ethyl]phenoxy]acetic acid (BMY 42393) > octimibate > CU 23 (a novel diphenylindole). From IP receptor binding affinities obtained on human platelet membranes, it is suggested that the slightly shallower log concentration-response curves for BMY 45778, BMY 42393, and CU 23 may reflect the near-maximal receptor occupancy required for complete relaxation. A fifth nonprostanoid, CU 602, had much shallower log concentration-response curves than cicaprost against phenylephrine tone but not against U-46619 tone; this may indicate IP receptor partial agonism coupled with TP receptor antagonism. The relaxant actions of the nonprostanoid mimetics were more persistent than those of the prostacyclin analogs on washout of the organ bath; by the inhalation route, this type of compound may be retained within pulmonary tissue and thus afford greater pulmonary/systemic selectivity than currently used pulmonary vasodilators.

The effect of the stable prostacyclin analog iloprost and its mechanism of action were investigated with the use of pressurized rat tail small arteries with a spontaneous myogenic tone. Iloprost concentration dependently dilated these vessels with a half-maximal effective dose of 5.0 +/- 0.5 x 10(-8) M. Application of 10(-7)-10(-6) M glibenclamide, a blocker of ATP-sensitive potassium (K(ATP)) channels, inhibited the iloprost-induced dilation. Glibenclamide did not affect the basal vessel diameter. The application of 5 x 10(-5)-10(-3) M tetraethylammonium (TEA) and 5 x 10(-9)-10(-7) M iberiotoxin, blockers of calcium-activated potassium (K(Ca)) channels, decreased vessel diameter in the presence of iloprost. Both TEA and iberiotoxin reduced the basal vessel diameter. Glibenclamide at 10(-6) M inhibited the dilation produced by 5 x 10(-5) M Sp-5,6-DCl-cBIMPS, an activator of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. Iberiotoxin at 10(-7) M decreased vessel diameter in the presence of Sp-5,6-DCl-cBIMPS. H-89 and Rp-8-CPT-cAMPS, blockers of cAMP-dependent protein kinase A (PKA), inhibited the iloprost-induced dilation of these vessels. With use of the whole cell configuration of the patch-clamp technique, it was observed that 5 x 10(-7) M iloprost enhanced an outward current, determined largely by K(Ca) channels, 1.79 +/- 0.17-fold in freshly isolated smooth muscle cells from rat tail small artery. These data show that iloprost dilates rat tail small arteries with a spontaneous myogenic tone and suggest that K(ATP) as well as K(Ca) channels are involved in this effect, which is mediated, at least partly, by PKA.

The patch-clamp technique was used to investigate the effect of iloprost on activity of calcium-activated potassium (KCa) channels of freshly isolated rat tail artery smooth muscle cells. In the whole cell configuration, outward current, determined largely by KCa channels, was enhanced 1.73 +/- 0.11-fold by 5 x 10(-7) M iloprost, 1.80 +/- 0.12-fold by 10(-4) M 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-cyclic monophosphothioate (Sp-5,6-DCl-cBIMPS), a specific activator of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), and 2.78 +/- 0.95-fold by 10 U/ml of the catalytic subunit of PKA + 10(-4) M MgATP, whereas the heat-inactivated catalytic subunit of PKA + MgATP was without effect. Iloprost at 5 x 10(-7) M increased this current 1.70 +/- 0.27-fold after pretreatment of cells with 10(-6) M okadaic acid, a specific phosphatase inhibitor, but did not alter this current after pretreatment of cells with 2 x 10(-4) M Rp-8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate, a specific PKA inhibitor. In the cell-attached configuration, activity of KCa channels was enhanced 2.48 +/- 0.44-fold by 5 x 10(-7) M iloprost and 2.09 +/- 0.07-fold by 10(-4) M Sp-5,6-DCl-cBIMPS. Iloprost at 5 x 10(-7) M did not alter intracellular calcium concentration in these cells measured using indo 1. In the inside-out configuration, activity of KCa channels was increased 87.12 +/- 45.04-fold by 10 U/ml of the catalytic subunit of PKA together with 10(-4) M MgATP, whereas no effect was observed after application of the catalytic subunit of PKA together with its regulatory subunit and MgATP, MgATP, cAMP, or the catalytic subunit of PKA alone also did not change KCa channel activity. Thus these results show that iloprost is able to activate KCa channels of freshly isolated rat tail artery smooth muscle cells and suggest that this effect is mediated by a PKA-induced phosphorylation of the channel.

In the isolated guinea pig aorta, the prostaglandin I2 analog iloprost (0.01-10 microM) inhibited the contractions induced by the thromboxane A2 analog U46619 (9,11-dideoxy-11 alpha,9 alpha-epoxymethanoprostaglandin F2 alpha; 30 nM) and prostaglandin F2 alpha (PGF2 alpha, 1 microM) in a concentration-dependent manner. In contrast, iloprost only partially inhibited the high K+ (65.4 mM)-induced contraction. In the muscle stimulated with high K+, verapamil (0.3 and 10 microM) inhibited [Ca2+]i and muscle tension in parallel, whereas iloprost (1 microM) inhibited muscle tension with only a small decrease in [Ca2+]i. In the muscle stimulated with U46619 (30 nM), verapamil and iloprost decreased both [Ca2+]i and muscle tension. However, as compared with the effect of verapamil, iloprost more strongly inhibited muscle tension than [Ca2+]i. The iloprost (0.1-1 microM)-induced relaxation was accompanied by a concentration-dependent increase in cAMP content. It was further demonstrated that inhibition of the U46619-contractions was augmented in the presence of cycloxygenase inhibitors, such as indomethacin (10 microM), ibuprofen (10 microM) and aspirin (10 microM). In contrast, the inhibition of PGF2 alpha-induced contraction was not affected by indomethacin. Similarly, the inhibitory effect of forskolin on U46619-induced contractions, but not on PGF2 alpha-induced contraction, was enhanced by indomethacin. These results suggest that iloprost inhibits vascular smooth muscle contraction by decreasing [Ca2+]i and the Ca2+ sensitivity of contractile elements through a cAMP-dependent mechanism. The results also suggest that in U46619-stimulated muscle, vasoactive prostaglandins that counterbalance the relaxing action of cAMP may be generated.

It has been suggested that insulin exerts a vasodilating effect, but the mechanisms involved are not completely understood. Since cyclic nucleotides mediate the vasodilation induced by endogenous substances, such as prostacyclin and nitric oxide, we aimed to investigate the influence of insulin (concentration range 240-960 pmol/l) on both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) content in human vascular smooth muscle cells. Insulin dose-dependently increased both nucleotides (cAMP: from 0.7 +/- 0.1 to 2.6 +/- 0.4 pmol/10(6) cells, p = 0.0001; cGMP: from 1.3 +/- 0.2 to 3.4 +/- 0.7 pmol/10(6) cells, p = 0.033). This increase is receptor-mediated, since it was blunted when cells were preincubated with the tyrosine kinase inhibitor genistein. The effect of insulin remained significant (p = 0.0001) when preincubation with the phosphodiesterase inhibitor theophylline prevented cyclic nucleotide catabolism. The increase of cGMP was blunted when the cells were preincubated with the guanylate cyclase inhibitor methylene blue, and with the nitric oxide-synthase inhibitor NG-monomethyl-L-arginine. At all the concentrations tested, insulin potentiated the increase of cAMP induced by the stable prostacyclin analogue Iloprost (p = 0.0001), whereas only at 1920 pmol/l did it potentiate the cGMP increase induced by glyceryltrinitrate (p = 0.05). This study demonstrates that the vasodilating effects exerted by insulin may at least in part be attributable to an increase of both cGMP and cAMP via a receptor-mediated activation of adenylate and guanylate cyclases in human vascular smooth muscle cells and that the insulin effect on cGMP is mediated by nitric oxide.

1. Membrane potential and tension were measured simultaneously in ring segments of main coronary artery of guinea-pigs. The synthetic thromboxane A2 analogue U46619 depolarized the tissues from -58 +/- 2 to -40 +/- 1 mV and increased tension by 12 +/- 1 mN mm-1. Nitric oxide (NO) and Iloprost, the stable analogue of prostacyclin, evoked hyperpolarization and relaxation. 2. The concentration of NO required to evoke half-maximal hyperpolarization (EC50 of 2 x 10(-5) M) was 40-fold higher than that which was required to induce relaxation (EC50 of 5 x 10(-7) M). The EC50 for Iloprost-induced hyperpolarization (3 x 10(-8) M) was similar to that for relaxation (4 x 10(-8) M). 3. Glibenclamide (10(-6) M) abolished the hyperpolarization in response to both NO and Iloprost but was without effect on the amplitudes of the relaxations over the complete concentration-response curves. 4. Acetylcholine evoked concentration-dependent hyperpolarization and relaxation in the presence of N omega-nitro-L-arginine methyl ester (NAME; 10(-5) M) and indomethacin (10(-6) M), and these responses were attributed to endothelium-derived hyperpolarizing factor (EDHF). The hyperpolarization produced by EDHF always preceded relaxation, and relaxation never occurred at concentrations of acetylcholine that were insufficient to evoke hyperpolarization. 5. The concentration-hyperpolarization and concentration-relaxation curves in response to acetylcholine were not affected by glibenclamide or barium (1-3 mM) but were shifted to the right 4- and 5-fold, respectively, by 1 mM tetraethylammonium. The hyperpolarization and relaxation evoked by acetylcholine were also reduced in a parallel manner when the potassium concentration in the superfusate was increased. 6. Hyperpolarizing current steps, applied to spiral strips of coronary artery denuded of endothelium and depolarized and constricted with U46619, caused relaxation. The relationship between hyperpolarization and relaxation evoked electronically was similar to that which was due to EDHF in intact tissues stimulated with acetylcholine. 7. It is concluded that the ability of NO or Iloprost to relax guinea-pig coronary artery does not depend upon hyperpolarization of the smooth muscle. In contrast, hyperpolarization is likely to play a major, if not the only, role in the relaxation in response to EDHF in this tissue.

In addition to inhibition of platelet function, prostacyclin and its stable analogues are reported to attenuate vascular smooth muscle cell proliferation. However, desensitization of prostacyclin responsiveness is a known phenomenon in platelets. In this study we investigated the time-dependent effects of the prostacyclin-mimetic iloprost and of PGE1, respectively, on PDGF-induced proliferation of cultured coronary artery smooth muscle cells. Proliferation, assessed by [3H]thymidine incorporation was markedly inhibited by coincubation with iloprost (100 nM) and PGE1 (100 nM) for 4 h. In contrast, addition of iloprost (100 nM) for 24 h did not decrease smooth muscle cell proliferation, whereas inhibition by PGE1 or by forskolin was not diminished. These results suggest a homologous desensitization of anti-mitogenic effects of iloprost in coronary artery smooth muscle cells, probably at receptor-level.

The effect of indomethacin on dilator prostanoid receptor-mediated cAMP formation was investigated using primary cultures of vascular smooth muscle cells from the newborn pig cerebral microvessels. Cerebral microvascular smooth muscle cells responded to dilator prostanoids (iloprost > PGE2) by increasing cAMP formation and release into the media (EC50 = 2 x 10(-8) M and 2 x 10(-7) M for iloprost and PGE2, respectively). Indomethacin inhibited iloprost- and PGE2-evoked increases in cAMP formation (IC50 = 10(-4) M) and release (IC50 = 10(-6) M) by microvascular smooth muscle cells (maximal inhibition 80-90%), whereas isoproterenol-induced cAMP formation was only slightly attenuated at the highest concentration of indomethacin used (10(-3) M). Aspirin was much less effective in inhibiting dilator prostanoid-induced cAMP formation and release by the cells. Direct analysis of prostacyclin receptor sites using [3H]iloprost as the ligand revealed saturable, high affinity (ED50 = 2 x 10(-8) M) and reversible binding to the membranes isolated from cerebrovascular smooth muscle cells. Indomethacin dose-dependently inhibited [3H]iloprost receptor binding (ID50 = 10(-4) M; maximal inhibition, 70%). The present data suggest that combination of highly effective inhibition of prostaglandin H synthase and receptor binding resulting in inhibition of dilator prostanoid-mediated cAMP formation in target cells may contribute to the increased efficacy of indomethacin compared with other prostaglandin H synthase inhibitors in blocking certain vasodilator responses associated with prostanoids.

The isolated canine spleen was perfused at constant flow with continuous recording of splenic arterial perfusion pressure (SAPP) and spleen weight. Intra-arterial injections of the thromboxane A2 (TXA2) mimetic U46619 caused dose-related increases in splenic arterial perfusion pressure (SAPP) of short duration (ED50 0.31 nmol). There were very small changes in spleen weight accompanying any of the vasoconstrictor responses to U46619. The stable analogue of prostacyclin, iloprost, caused dose-dependent reductions in SAPP (ED50 1.3 nmol) indicating vasodilation. There were no changes in spleen weight to any doses of iloprost indicating a lack of action on capsular smooth muscle. Similarly, the nitric oxide (NO) mimetic sodium nitroprusside caused dose-related reductions in SAPP of short duration (ED50 5.8 nmol). No changes in spleen weight accompanied splenic vasodilator responses to any dose of sodium nitroprusside (SNP). The results indicate the potential actions and intrinsic potency of three endogenous vasoactive substances and provide information about their relative roles in the control of the splenic microcirculation in situations when they are released.

Cerebral vasodilation in response to hypercapnia involves prostanoids in newborn pigs. This study examines the hypothesis that endothelial injury in vivo inhibits cerebral vasodilation and prostacyclin synthesis in response to hypercapnia, thus suggesting prostacyclin is a primary endothelium-derived vasodilating factor in newborn pig cerebral circulation. Anesthetized piglets with closed cranial windows were studied before and after injury caused by light/dye or before and after dye-only sham control. Light/dye injury was produced by injecting sodium fluorescein intravenously and passing filtered light from a mercury arc lamp through the cranial window. Ultrastructural changes to endothelium of pial vessels were produced that were characterized by surface pits, vacuolar cytoplasmic inclusions, and mitochondrial injury. After the light/dye injury, dilation to hypercapnia was absent while dilations to iloprost, isoproterenol, and sodium nitroprusside and constrictions to norepinephrine and acetylcholine were retained. Before light/dye treatment, hypercapnia increased cortical periarachnoid 6-keto prostaglandin F1 alpha concentration approximately threefold. However, after treatment, 6-keto-prostaglandin F1 alpha was not increased significantly in response to hypercapnia. These findings are consistent with the hypothesis that endothelial prostacyclin synthesis induced by hypercapnia participates in dilation of adjacent smooth muscle.

Cellular injury is a major cause of intimal hyperplasia in vein grafts. The effects of exposure of vein samples to iopamidol, papaverine and iloprost were studied in vitro in an organ chamber to determine whether these agents cause endothelial and/or smooth muscle cell injury. Smooth muscle cell function was assessed by eliciting a dose response curve to noradrenaline. Endothelial cell function was assessed by measuring the degree of endothelial-dependent relaxation of sub-maximally contracted vein samples. Iopamidol and iloprost did not have any deleterious effect on endothelial or smooth muscle cell function. Papaverine did not affect endothelial-dependent relaxation but did produce a significant decrease in smooth muscle contraction. The use of these intraoperative agents during femorodistal bypass does not appear to cause functional injury to the endothelial or smooth muscle cells.

Effects of dexamethasone, retinoic acid, prostaglandin E2 (PGE2), and Iloprost as a agonist of prostacyclin (A-PGI2) on DNA synthesis and production of a precursor of matrix metalloproteinase 1 (tissue procollagenase/proMMP-1) by human aortic smooth muscle cells were investigated. When after treatment with platelet-derived growth factor (PDGF), these agents were added to the cultures, DNA synthesis and production of proMMP-1 were inhibited in a dose-dependent manner. These results suggest that these agents are negative regulators of PDGF. Since these agents are present in the blood or produced in the blood wall, in addition, since PDGF plays the most important role in the process of atherosclerosis, we propose that these agents function in vivo as a systems of protection against atherosclerosis.

Objective: To examine whether elevated intravascular pressure in chronic hypertension alters responses of vascular smooth muscle to agents of endothelial origin., Methods: Coarctation hypertensive, sham normotensive control, and one-kidney, one clip hypertensive (1K1C) rats were used. Tail systolic, carotid and femoral arterial pressures were measured. Responses to histamine, endothelin-1 and the prostacyclin analog iloprost were evaluated in isolated helically cut strips of thoracic and abdominal aortas, with and without endothelium, from all groups. Responses to nitroglycerin were also evaluated in strips of abdominal aortas., Results: Thoracic aortas from 1K1C and coarctation hypertensive rats, as well as abdominal aortas from 1K1C rats, but not abdominal aortas from coarctation hypertensive rats were exposed chronically to elevated arterial pressure. Endothelium-dependent maximal relaxation by histamine was significantly depressed in thoracic aortas from both groups of rats, as well as in abdominal aortas from 1K1C rats. Maximal relaxation and sensitivity to histamine were normal in abdominal aortas from coarctation hypertensive rats. Sensitivity to nitroglycerin was impaired in abdominal aortas from 1K1C rats but not in those from coarctation hypertensive rats; maximal relaxation to nitroglycerin was similar in all groups. Relaxation to iloprost was independent of the endothelium, observed only in thoracic aortas and impaired in hypertensive rats. Responses to endothelin-1 were similar in the groups., Conclusion: Vasorelaxation by histamine, iloprost and nitroglycerin are impaired in hypertension. The impaired relaxation by histamine results from exposure of the vascular endothelium to chronically elevated pressure. This impairment may be related to effects of high pressure in reducing the ability of the endothelium to produce endothelium-derived relaxing factor and inhibit cyclic GMP-dilator mechanisms.